Mutagenic studies that implicate specific amino acids in reaction chemistry or in substrate binding are fully interpretable only when the structural integrity of the variant protein has been validated. This validation is facilitated by identification of appropriate spin-labeled substrate analogs. Measurement of spin-probe affinity, binding stoichiometry, and correlation time using D42A and D169A mutants and comparison of these parameters with those measured for wild-type protein affords an elegant, rigorous, yet efficient solution-state method for validating the structural integrity of engineered proteins. A spin-labeled ATP analog (ATPSAP) is being productively used to validate the integrity of the engineered variants D42A and D169A of prokaryotic phosphoribulokinase which catalyzes a key step in carbon dioxide assimilation in Calvin's reductive pentose phosphate cycle.